The present invention relates to the field of fungal biotechnology, more particularly to genetic engineering methods for the production of carotenoids in fungal hosts selected from Rhodospordium and Rhodotorula genera.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
It is well documented that carotenoid production is initiated with the biosynthesis of geranylgeranyl diphosphate (GGPP) catalyzed by GGPP synthase for the condensation of C15 farnesyl diphosphate (FPP) and C5 isopentenyl diphophate (IPP). Subsequently, two molecules of GGPP are further condensed to form the colorless precursor phytoene, which is catalyzed by phytoene synthase. In fungi and eubacteria, phytoene desaturase catalyzes all 4 steps of desaturation of phytoene to yield the red colored lycopene while this is done by separate phytoene desaturase and γ-carotene or turase and desaturase in plant, algae and cyanabacteria. Lycopene is cyclized by carotene cyclase to form mono-cyclic γ-carotene and teneene cy, and dicyclic α-carotene and carotene a [1, 2]. Further upstream of the biosynthetic pathway, FPP is produced by farnesyl diphosphate synthase (FPS) catalyzed condensation of IPP and C10 geranyl diphosphate (GPP), the latter produced by GPP synthase catalyzed condensation of IPP and dimethylallyl diphosphate (DMAPP), the product of IPP isomerase IPP and DMAPP can be synthesized via either the mevalonate pathway (MVP) or 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway (MEP/DOXP) [3, 4].
Carotenoids are 40 carbon (C40) tetraterpenoids [5]. The unoxygenated carotenoids, such as γ-carotene, β-carotene and lycopene are known as carotenes. Further enzymatic modifications of carotenes produce molecules containing oxygen, such as lutein, retinol (vitamin A), zeaxanthin and astaxanthin [6, 7]. Biosynthesis of carotenoids occur in all photosynthetic organisms [8] and many non-photosynthetic microorganisms, such as bacteria and fungi [1, 5, 9, 10], and some insects [11].
Carotenoids play important role in human and animal health and development [12-15]. For example, a higher dietary intake of carotenoids was associated with a lower risk for age-related macular degeneration (AMD) [13]; vitamin A deficiency is associated with abnormal growth of the skeleton and teeth and infertility in rat [14]; retinal (retinaldehyde) is essential for vision while retinoic acid is essential for skin health, teeth remineralization and bone growth [16]; intakes of lycopene is related to lower risk prostate cancer [17]. Carotenoids are natural colorant with many colors available [18-20]. Carotenoids are precursors for the production of valuable aromatic compounds [21]. β-carotene can be cleaved by P450 cytochrome oxidase to make retinal (retinaldehyde) [16], which is essential for vision and when converted to retinoic acid, it is essential for skin health, teeth remineralization and bone growth. Therefore, carotenoids are valuable food and feed additives, neutraceuticals and cosmoceutical.
Retinol, retinal and retinoic acid are known as retinoids, which are derived from breakdown of skin health, teeth remineralization and bone growth. Retinol and retinal is interconvertable and catalyzed by alcohol dehydrogenase and short-chain dehydrogenase/reductases whereas aldehyde dehydrogenase and cytochrome P450 enzyme families catalyze the irreversible oxidation of retinal to retinoic acid. The identification of enzymes catalyzing retinol oxidation in vivo has been controversial, in part due to the difficulty by the reversible nature of this reaction [22].
Rhodosporidium and Rhodotorula are two fungal genera belonging to the Pucciniomycotina subphyla. They can be cultured in single-cell form in very high cell density in fermentors at a fast growth rate and accumulate high levels of triacylglyceride [23-26]. Rhodosporidium and Rhodotorula are able to produce high levels of carotenoid [27-30], with beta-carotene, gamma-carotene, torularhodin and torulene being the major components [31]. Torularhodin and torulene are potential colorants and inducer of gene expression. Apart from the identification of a putative CAR2 homolog [32], there is no report on the carotenoid biosynthetic pathway in Rhodosporidium and Rhodotorula. Any method that improves the productivity and product purity of carotenoids and their derivatives are of commercial value and significance.